IMMUNOHEMATOLOGY (BLOOD BANKING) – RMT 2024

LEC 9: ANTIHUMAN GLOBULIN TESTING

Eunice Fay Cayacap, RMT, MLS (ASCPi)CM
March 2023

INTRODUCTION MONOSPECIFIC AHG

• Most clinically significant blood group antibodies fall into the IgG or • Should only contain one antibody specificity
IgM immunoglobulin classes with occasional IgA forms found among • Either: Anti-IgG OR antibody to specific complement components
autoantibodies and against antigens in certain blood group systems (e.g C3b/C3d).
• There is little to no problem in the detection of IgM due to its large
pentameric structure and thus, it can react to a corresponding antigen Upon performing the Direct Antiglobulin Test (DAT) with a
and agglutinate red cells suspended even in saline. polyspecific AHG, it should be tested further with a monospecific
• On the other hand, IgG antibodies are termed incomplete or non- reagent because this additional testing will provide further
agglutinating antibodies due to its monomeric structure, thus, it can information in determining the type of immune hemolytic anemia
only cause sensitization and cannot directly agglutinate the red cells. involved.
• The specific antihuman globulin antibodies that will act as a bridge will
induce agglutination with these erythrocytes coated with human I. ANTI-IgG
immunoglobulin and/or complement.
• NO complement activity
ANTIHUMAN GLOBULIN TEST → Should not be able to react with cells coated or sensitized with
any complement protein
• May also be referred to as the Coomb’s Test • Contains antibodies specific for the Fc or the crystallizable fragment
of the gamma heavy chain of the IgG molecule
PRINCIPLE • However, if it is not labeled as heavy chain specific, it may contain
light chain specificity to allow the detection of IgM and IgA antibodies
• Antihuman globulins (AHGs) obtained from immunized nonhuman • Light chain specificity: IgM, IgA, IgG
species bind to human globulins such as IgG or complement, either
free in serum or attached to antigens on red blood cells (RBCs). II. ANTICOMPLEMENT
• The antiglobulin will produce a bridge between the RBCs by
employing a second antibody that will react with the first antibody or • Reactive only against the designated complement components and
the complement attached to the red cell. contain NO activity against human immunoglobulins
→ This bridge will now span the gap between the red cells and will • Monospecific anticomplement reagents can still be a blend of
produce an agglutinate. monoclonal anti-C3b and monoclonal anti-C3d.
HISTORY
• Polyclonal and Monoclonal: refers to the preparation or how the
AHG or how these antibodies are being prepared in the laboratory; the
• In 1908, Moreschi described the principle of the test. He used rabbit
method of preparation
antigoat serum to agglutinate RBCs that were sensitized with low
non-agglutinating doses of goat antirabbit RBC serum. • Polyspecific and Monospecific: refers to the specificity of these
antibodies
• In 1945, Coombs and associates described the use of the antiglobulin
test for the detection of weak and non-agglutinating Rh antibodies in
serum. PREPARATION OF AHG
→ Before the antiglobulin test was developed, blood bankers were
only able to detect IgM antibodies.
→ With the help of AHG, IgG antibodies can now be detected as
well.
• In 1946, Coombs and coworkers described the use of AHG to detect
in vivo sensitization of the RBCs of babies suffering from hemolytic
disease of the newborn (HDN).
• In 1947, Coombs and Mourant demonstrated that the antibody • The classic method of AHG preparation involves injecting human
activity that detected Rh antibodies was associated with the anti- serum or purified globulin into laboratory animals such as rabbits
gamma globulin fraction in the reagent. • May be produced by hyperimmunizing animals usually rabbits with
• In 1951, Dacie presented the first indication that there might be purified immunoglobulin or complement to produce high-titered, high-
another antibody present that influenced the final reaction. avidity IgG and complement antibodies
→ Was able to observe that different reaction patterns were obtained • Example: If we Inject a human IgG to a rabbit, it will produce anti-IgG
when dilution of the AHG were used to test cells sensitized with • Human complement components injected into a rabbit will result to the
warm as compared with cold antibodies. production of anti-complement
• In 1957, Dacie and coworkers published data showing that the → Because these human complement components are antibodies
reactivity of AHG to cells sensitized with warm antibodies resulted will move or react as a foreign antigen to the rabbit
from anti-gamma globulin activity whereas anti-non gamma → It is an antigen to the rabbit, thus the rabbit will produce an
globulin activity was responsible for the activity of cells sensitized by antibody against that antigen. In that case, it’s anticomplement
cold antibodies. and anti-IgG.
→ The non-gamma globulin component was shown to be a β
globulin and had specificity for the complement which was later I. POLYCLONAL ANTIBODIES
revealed that the complement activity was a result of C3 and C4.
• A mixture of antibodies from different plasma cell clones
AHG REAGENTS • Recognize different antigenic determinants (epitopes)
POLYSPECIFIC AHG
II. MONOCLONAL ANTIBODIES
• Also known as the broad-spectrum AHG
• Contains antibody to human IgG and to the C3b/C3d component of • Derived from one clone of plasma cells
human complement • Recognize a single epitope in an antigen
• RBC auto and alloantibodies can be occasionally IgA or a mixture of
each immunoglobulin POLYCLONAL AHG PRODUCTION
→ To detect reactions with IgA or IgM, the polyspecific AHG may
have antibody activity to the kappa or lambda light chains. • Conventional polyspecific antiglobulin reagents are produced by
• Facilitate agglutination when RBCs have been sensitized with IgG or immunizing one colony of rabbits with human immunoglobulin (IgG)
C3d or both. antigen, and another colony with human C3 antigen.
→ anti-C3b, anti-C4b, anti-C4d • Because of the diversity or the diverse characteristic of the IgG
→ anti-IgA, anti-IgM molecule, the use of serum from many donors to prepare the pooled
IgG antigen used to immunized the rabbits if being employed
• After immunization of the rabbits, we extract blood sample from them
and pooled anti-IgG is also derived so that we can produce a
polyclonal antibody that can recognize the different kinds of epitopes
of the IgG molecule
• No matter how unique the IgG antibody is, it can still be detectable
with the use of the polyclonal antibody because it targets many
epitopes in a single antigen.
• Uses serum from many donors to produce polyclonal reagents that
are capable of detecting many antibodies.

Immunohematology (Blood Banking), CDU – BSMT3 Page 1 of 9

• Both colonies are hyperimmunized to produce high titer, high-avidity
IgG antibodies.
→ After both colonies are hyperimmunized with human C3 and
human immunoglobulin antigen, the animals are then bled(?) if
the antibody potency and specificity meet the predetermined
specification; followed by absorption with A1, B, and O cells.
→ The total antibody content of the pool is then measured and
potency is analyzed by performing block titrations.
• Block titrations:
→ Anti-IgG
▪ Done by reacting dilutions of each antibody against cells
sensitized with different amounts of IgG.
▪ Very important to prevent false-negative results due to
prozone phenomenon— antibody excess.
→ Anti-C3
▪ Potency is measured using at least two examples each of a
C3b- and C3d- coated cells.

NOTE:
Take note of the difference:
In anti-IgG block titration, the cells are sensitized with a pre-
measured amount of IgG. Since it is not possible to coat red cells
with a measured amount of complement, we only make use of at
least two examples of C3b- and C3d- coated cells regardless of
their amount in a red blood cell or a cell.

• This is the hybridoma technology used in the production of monoclonal

antihuman globulin.
→ We have a population of mice injected with purified human
antigen—either IgG or complement.
→ The mouse spleen cells will then be collected or removed so that
we will fuse it with myeloma, a non-antibody secreting cell.
→ Now, the spleen cells of the mice plus the myeloma cell will
yield or will result to form the hybridoma.
(mice spleen cells + myeloma cell = hybridoma)
→ This hybridoma will grow into the tissue culture to yield the
monoclonal antibodies, so that it will become the monoclonal
anti-IgG or the monoclonal anti-C3b, -C3d, and -C3g.

AHG REAGENTS

• Do not be confused, polyspecific AHG can be made

using polyclonal or monoclonal antibody.
• Even though it's polyspecific, it can be polyclonal or
monoclonal in origin.
• The monospecific AHG only has one antibody
specificity, but it can be produced as a monoclonal-
polyclonal formula.
• Before the AHG reagent is available for purchase in the
market, manufacturers must subject their agents to
• Polyclonal AHG production will start by injecting an immunogen into the evaluation procedures, and the results must be
animal so there is one pool for IgG and one for complement. submitted to the Food and Drug Administration for
• This will induce the production of high levels of antibody into the blood of approval.
rabbits.
• Meaning, the activity of the two components will always vary
NOTE: somewhat among different manufacturers of the reagent and even
between lots produced by the same manufacturer, BUT all AHG sera
If we are requiring large volumes of antibody, sheep or goats may must contain levels of anti-IgG and anti-C3d activity that will meet
be used or even exceed the reference standards for the Food and Drug
administration.
MONOCLONAL AHG PRODUCTION • AHG reagent should also contain buffers, stabilizers, and
bacteriostatic agents, and most importantly it may be dyed colored
• Devised by Kohler and Milstein. GREEN for identification purposes.
• Usually begins with immunization of laboratory animals, usually
mice, with a purified human globulin.
→ After a suitable immune response has been established, the
mouse spleen cells are fused with myeloma cells.
(Myeloma cells are non-secreting antibody cells so they are fused
with mouse spleen cells.)
→ After the fusion, we have the resulting hybridomas which are
screened for antibodies with the required specificity and affinity.
→ The antibody-secreting clones may then be propagated in tissue
culture or by inoculation into the mice. In which case, the
antibody is collected as ascites.
• ALL antibody molecules produced by a clone of hybridoma cells are
already identical in terms of antibody structure and antigen
specificity
→ There is no need for absorption with A1, B, and O cells to
remove heterospecific antibodies.

These are the several AHG reagents defined by the FDA Center for
Biologics Evaluation and Research. (REFER LAST PAGE)

Immunohematology (Blood Banking), CDU – BSMT3 Page 2 of 9

 ANTIBODIES REQUIRED IN AHG A. DIRECT ANTIGLOBULIN TEST (DAT)
I. Anti-IgG PRINCIPLE AND APPLICATION

• Even with the use of enhancing reagents and the final step of Detects IN VIVO sensitization of RBCs with IgG or complement
centrifugation, many IgG antibodies still cannot produce any components.
detectable agglutination. • A very important serologic panel (one step test).
→ To visualize these reactions by these non-agglutinating and
incomplete antibodies, AHG sera with IgG specificity must be Application:
utilized. • Hemolytic disease of the newborn (HDN)
• Majority of these anti-IgG antibodies are a mixture of IgG1 and IgG3 → Result: POSITIVE
subclasses. → maternal IgG antibodies that will cross the placenta will attach to
• IgM = may be detected by anticomplement the fetal erythrocytes.
→ This is rarely encountered. However, since these antibodies are • Hemolytic transfusion reaction (HTR)
the ones most efficient in activating the complement, they can still → Result: POSITIVE
be detected using the anti-complement reagent of AHG. If the → IgG/C3d or both and depends on the antibody responsible, first
anti-IgG is not heavy chain specific then the presence of any anti- immuno-hematologic evidence of a hemolytic reaction.
light chain activity will allow us for the detection of all ▪ Donor red cells have become coated with the recipient antibody.
immunoglobulin including IgA and IgM. • Autoimmune hemolytic anemia (AIHA)
• Presence of anti-light chain activity allows detection of all → Result: POSITIVE
immunoglobulin classes. → Most warm autoimmune antibodies can cause a positive DAT
result because of IgG antibodies and rarely because of both IgG
II. Anticomplement and C3d.
→ Most Warm Autoimmune Hemolytic Anemia is caused by IgG
• Some antibodies will also fix complement components to the red cell antibodies.
membrane after complex antigen-antibody. → For Cold Autoimmune Hemolytic Anemia:
• Improve reactions of clinically significant antibodies then in antibodies ▪ Cold autoagglutinins often cause a positive DAT result because
specific reagents was introduced. of C3d only.
• Indicating the need for anti complement activity in AHG to allow the • Drug-induced hemolytic anemia
IAT to detect antibodies, polyspecific reagent was introduced. → Result: POSITIVE
• Anti-C3c = MOST important anti complement component because of → because of IgG, C3d, or both depending on the mechanism and
its non-specific reactions. drug involved.
→ However, it is NOT commonly used because its amount is
NOTE:
decreased upon incubation with RBC longer than 15 minutes.
→ Anti-C3b was implemented to be used instead. DAT is NOT a required test in routine pretransfusion protocols.

DAT PANEL

• If initial DAT testing with a polyspecific antisera is POSITIVE, it may

be subjected to further testing with a Monospecific anti-IgG & anti-
C3d.
→ To determine the specific type of globulin causing the reaction
• Warm AIHA & transfusion reaction workup = red cells may be coated
with IgG or C3d or both.

This table is a Direct Antiglobulin Test result using an EDTA specimen. Assuming
in the polyspecific AHG sera, the patient already tested positive, further testing is
done using the Monospecific anti-IgG and anti-C3d. (table is combined from
references: Harmening & Turgeon)
PRINCIPLES OF THE ANTIGLOBULIN TEST
• Important in establishing the immune origin and the type of hemolytic anemia
The Antiglobulin test is based on the following simple principles: that the patient is manifesting because each type of hemolytic anemia is treated
differently.
1. Antibody molecules and complement components are globulins
2. Injecting an animal with human globulin stimulates the animal to
produce antibodies to the foreign protein. EVALUATION OF A POSITIVE DAT
• Our antibodies and complement components are considered as • The American Association of Blood Bank’s Technical Manual states
antigens once they are injected to our test animals (rabbit, mice, that a positive DAT test alone is NOT diagnostic. Meaning, the
sheep, and goats). interpretation of a positive DAT would require knowledge and
information on the patient’s diagnosis, drug therapy, and any recent
transfusion history.
• Answering these questions before investigating a positive DAT for
patients will help determine what further testing is appropriate.
1. Is there evidence of in vivo hemolysis?
→ Typically elevations of Bilirubin and even Reticulocyte
Count
2. Has the patient been transfused recently? If so, did the patient
receive blood products or components containing ABO-
incompatible plasma
→ Ask or look at previous records and you saw that the patient
have received blood products and components containing
ABO incompatible plasma
3. Was the patient able to undergo an antibody screen?
4. Does the patient's serum contain unexpected antibodies?
5. Is the patient receiving any drugs?
6. Is the patient receiving anti lymphocyte globulin or anti-thymocyte
globulin?
7. Is the patient receiving intravenous immune globulin or
intravenous Rh immune globulin?
8. Has the patient received a marrow or other organ transplant?

Immunohematology (Blood Banking), CDU – BSMT3 Page 3 of 9

 NOTE: FACTORS AFFECTING ANTIGLOBULIN TESTING
We have to take all of these into consideration before further testing
A. Ratio of serum to cells
B. Reaction medium
B. INDIRECT ANTIGLOBULIN TEST (IAT) C. Temperature
D. Incubation time
Determine IN VITRO sensitization of RBCs and is used in the following E. Washing of RBCs
situations: F. Saline for washing
• Detection of incomplete (nonagglutinating) antibodies to potential G. Addition of AHG
donor RBCs (COMPATIBILITY TESTING) or to screening cells H. Centrifugation for reading
(ANTIBODY SCREEN) in serum.
→ In here, reagent red cells of a known phenotype is incubated with A. RATIO OF SERUM TO CELLS
the patient’s serum to detect patient antibodies that have been
bounded to the reagent red cells either polyspecific or • MINIMUM of 40:1
monospecific antiglobulin reagent used. → Can be achieved by using 2 drops of serum and 1 drop of a 5%
• Determination of RBC phenotype using KNOWN antisera suspension of cells
→ Example of an RBC Phenotype that requires Antiglobulin Testing → Keep in mind that increasing serum to red cell concentration will
is Weak D. also increase the rate or affect the equilibrium constant of the red
→ Detection of many erythrocyte antigen will require an incubation cells sensitization of the test system
at 37 C with a specific antisera followed by conversion to an • Cells suspended in saline:
Antiglobulin Test. → 4 drops serum, 1 drop 3% cells in saline suspension
• Titration of incomplete antibodies → if cells suspended in saline is used, it is recommended to increase
the ratio of serum to cells to detect weak antibodies.
NOTE:
B. REACTION MEDIUM
Do not forget that RBC Phenotyping example is Weak D typing
which employs Indirect Antiglobulin Test
Albumin
• Allows antibody-coated cells to come into closer contact with each
other so that aggregation could occur.
• It will reduce zeta potential to permit RBCs to approach each other
and increase the chances of agglutination.
• Incubation period: 15-60 minutes

Low Ionic Strength Solutions

• Reduce zeta potential surrounding an RBC and increases the uptake
of an antibody into the RBC during sensitization phase which will
increase the possibility of agglutination.
• May be achieved by either suspending RBCs in LISS or using LISS
additive reagent.
• It is recommended that only 2 drops serum and 2 drops of 3% cell
suspension in LISS should be added.
• If you increase the serum-to-cell ratio using LISS, it may increase the
ionic strength of the reaction mixture that would lead to the decrease
of the sensitivity of the test medium and counteract the shortened
incubation time.
• Incubation period: 10-15 minutes (reduced incubation period)

Polyethylene Glycol
• MORE sensitive than LISS, Albumin and other saline system reaction
mediums.
→ But, in patients with elevated levels of plasma proteins, PEG is
not recommended due to an increase in the precipitation proteins
in the reaction medium
• Increases RBC sensitization by removing water molecules
surrounding the RBC to concentrate the antibody.
→ By removing the water, antibodies will be more concentrated
• Anti-IgG = AHG reagent of choice with PEG testing to avoid false
positive reactions.

NOTE:
Potentiators may be added to the test mixture before the 37 degree
celsius phase to increase the sensitivity of the test system. It will help
to have a shortened incubation period or incubation time.

C. TEMPERATURE

• The rate of reaction for the majority of IgG antibodies is optimal at

• IAT is a two-step procedure because we incubate the test mixture. 37°C and this is also the optimum temperature for complement
→ We incubate the red cells with the antisera because we give time for the activation.
antibody molecule attachment to the RBC antigen in case it was not able • For IgM antibodies, the temperature of the reaction environment may
to attach during the Immediate Spin or Saline Phase. be decreased to 24°C or 4°C because it is a cold-reacting antibody.
→ After incubation we perform a minimum of 3 saline washes that is to remove • To not miss any unwanted antibodies, the IgM antibodies should also
free globulin molecules. Why do we have to remove these free molecules? be given the optimum temperature to react.
Because these will neutralize the AHG reagent causing a false negative • Effects of temperature may affect both the equilibrium constant and
result.
the rate of reaction between the antigen and the antibody.
→ After a minimum of 3 washes, we add the antiglobulin reagent which will
suppose to be the bridge of these red cell agglutinates.
→ After, we centrifuge to accelerate the agglutination by bringing the cells
D. INCUBATION TIME
closer together.
→ Examine for agglutination. Interpret as positive or negative. Positive if • Different antigen-antibody reactions will reach equilibrium over varying
there is any form of agglutination of hemolysis in the test mixture. time periods.
→ Grade the agglutination reaction to determine the strength of the Ag-Ab • Majority of clinically significant antibodies can be detected after 30
complex. minutes of incubation.
→ To a negative IAT, always add antibody-coated RBCs. Antibody-coated • Extended incubation is rarely performed.
RBCs refer to Check Cells. Check cells are only added to a negative result. • Incubation time for cells suspended in saline may vary between 30
This will check for the neutralization of antisera by the free globulin
and 120 minutes.
molecules. This will validate the test result.
• When incubated in LISS or PEG, incubation time may be shortened
to 10 to 20 minutes.
→ Extended incubation even up to 40 minutes for the LISS have
been shown to cause antibody to elute from the red cells,
decreasing the sensitivity of the test.

Immunohematology (Blood Banking), CDU – BSMT3 Page 4 of 9

E. WASHING OF RBCs MODIFIED AND AUTOMATED
ANTIGLOBULIN TEST TECHNIQUES
• Washing of red cells is one of the most important and critical steps in
performing antiglobulin testing. • Low ionic polybrene technique
• RBCs must be saline-washed a minimum of three times before • Enzyme-linked antiglobulin test
adding the AHG reagent. • Solid phase technology
• Removes free unbound serum globulins. • Gel test
→ FALSE NEGATIVE – neutralization of the AHG reagent by
residual unbound serum globulins. A. LOW IONIC POLYBRENE TECHNIQUE
• Washing of cells can be controlled using check cells.
→ Check cells - Group O cells sensitized with IgG. • Relies on low ionic conditions to rapidly sensitize cells with antibody
• Washing should be performed IMMEDIATELY after being removed • POLYBRENE = a potent rouleaux-forming reagent, added to allow the
from the incubator and in as short a time as possible to minimize sensitized cells to approach each other and permit cross-linking by the
elution of low-affinity antibodies. attached antibody
• All saline is discarded after the final wash. • A high ionic strength solution is then added to reverse the rouleax; but
→ Centrifugation at each wash should be sufficient so that the cell it will REMAIN if TRUE AGGLUTINATION is present in the test
pellet will be firm and stable at the bottom. mixture
→ To minimize the possible loss of cells upon decantation of • If low ionic polybrene technique is performed a Monospecific anti-IgG
supernatant. MUST be used because the low ionic condition can cause
considerable amount of C4 and C3 to cope the cells and give false
F. SALINE FOR WASHING positive reactions if a polyspecific reagent is to be used.

• Saline used for washing should be fresh. B. ENZYME-LINKED ANTIGLOBULIN TEST

• Should be buffered to a pH of 7.2 to 7.4 for optimum antigen-antibody
reactions. • An RBC suspension is added to a microtiter well and washed with
→ Saline stored for long periods in long plastic containers has been saline
shown to have a decreased pH causing false negative results. • AHG, labeled with an enzyme is added to the microtiter well which
→ FALSE NEGATIVE = ACIDIC contains the RBC suspension
→ FALSE POSITIVE = BACTERIAL CONTAMINATION IN SALINE • AHG which has been labeled with the enzyme will bind to IgG-
sensitized red cells
G. ADDITION OF AHG → Excess antibody is removed
→ Enzyme substrate is added
• Color of AHG reagent: green • This is a kind of spectrophotometric determination because the
• Addition of AHG should be performed immediately after washing amount of color produced is measured spectrophotometrically and
to minimize the chance of antibody elution and subsequently it is proportional to the amount of the antibody present
neutralizing the AHG reagent. • The optical density is usually measured at 405 nm
• Addition of two (2) drops of AHG may overcome washing problems • The number of IgG molecules per red cells can also be
when low levels of serum remain in the test mixture. determined from this procedure

H. CENTRIFUGATION FOR READING C. SOLID PHASE TECHNOLOGY

• Agglutination is enhanced by centrifugation as antibodies • One of the test reactants is bound to a solid support and that depends
sensitize the erythrocytes, bringing them closer together. on the kind of test that the MT is performing.
• Center for Biologics Evaluation and Research (CBER) • Direct Coomb’s test:
recommendation: 1000 RCF for 20 seconds. → Antibody is attached to a microplate well (solid support), and
• Use of higher RCF will yield a more sensitive result because the RBCs are added
pellet is more intact at the bottom. → Serum and red cells are added to the direct and indirect coombs
• Optimum centrifugation conditions are determined for EACH individua test respectively to the solid support of the microplate
centrifuge in blood bank laboratories. well followed by an enhancement reagent
→ This test system is then incubated at 37C to allow binding of
SOURCES OF ERROR the antigen and antibody, We then wash the entire system to
remove any unbound serum globulins. Followed by the addition
of RBC coated with anti-human IgG then the plates are
centrifuged
→ (+) : The bottom of the well will be covered with suspension
→ (-) : RBCs will settle to the bottom of the well
• Indirect Coombs’ test:
→ Known RBCs are bound to a well treated with glutaraldehyde or
poly L-lysine
Solid Phase Test

familiarize these sources of error, false-positive and false-negative results

especially those that not included in the notes I gave you for the laboratory
activity. REFER LAST PAGE for clear sources of error

NEW TECHNOLOGIES AND AUTOMATION

IN BLOOD BANK TESTING

• Throughout the history of immunohematology as a clinical discipline,

manual agglutination tests have always been the most popular
method employed for various processes
• In general, blood banks have landed in other areas of the clinical
laboratory when it comes to the automation of the manual method
• The conventional manual agglutination method was first automated
only in large blood centers for performing ABO and Rh donor typing
but, now we have few or a lot of automated techniques employed in
blood banking

Side view of Microplate

Immunohematology (Blood Banking), CDU – BSMT3 Page 5 of 9

 REMEMBER:
Gel test is still based on the principle of heme agglutination (antigen-
antibody reaction)

This is a homogenous suspension of the red cells

REMEMBER:
If POSITIVE then the bottom well will be covered with a suspension
If no specificity occurs, RBCs will only settle at the bottom well

Picture of an entire microtube

Upper, wider part - reaction chamber
Lower, narrow part - gel column
• RBCs, serum or plasma is allowed to incubate in the reaction chamber.
• Following incubation and controlled centrifugation conditions, the red cells will
bind to the corresponding antibody (the reaction happens in the reaction
chamber)

Comparison of a Grading Chart of the conventional tube technique (left) and solid
phase technology (right)
Take note of the equivalent reactions:
• Negative result in conventional tube technique (no agglutination) = discreet
button in the bottom of the well indicating negative reaction in the solid phase • For testing using the gel technology or gel card, measured volumes of serum or
technology (bottom most: 0 grading) plasma and red cells are dispensed into the microtubes reaction chamber where
• One solid agglutinate in conventional tube technique = bottom of the well in solid the red cells are sensitized or it is where the antigen-antibody binding happens
phase technology will be covered with the cell suspension (topmost: 4+ grading) (usually during incubation period)

• The wells on the right (solid phase technology) can be read manually or with the
help of an automated reading device
• Positive reactions are very stable and can be read up to 2 days later
• These plates can be covered and stored at temperatures of 2-8C
• However, there is also a disadvantage: a need for specialized equipment
→ Microplate centrifuge
→ Specialized 37C incubator (for microplates)
→ Light source is also sometimes needed for reading of the final result

D. GEL TEST

• Gel technology • The gel card has its own incubator and the temperature is always set at 37C
• Discovered in 1985 by Dr. Yves Lapierre when he was trying to • Following 37C incubation period, the gel card is spun in a specialized centrifuge
investigate ways on how to trap red blood cell agglutinates under controlled conditions
• Detect RBC antigen-antibody reactions by means of using a chamber
filled with gel • Major Disadvantage: The need to purchase these special
→ The gel is made up of polyacrylamide gel or dextran acrylamide equipments
gel and will serve as a trap → Centrifuge, incubator, containment rack that will accommodate
• The free unagglutinated cells do not have any problem travelling these microrube cards used for testing
through the length of the column to form a pellet at the bottom of the → Sometimes, you may even need specialized pipette
tube • Major Advantage: Standardization
→ (-) : free, unagglutinated RBCs form pellets in the bottom of the → No tube shaking that will resuspend the red cell button because
tube the tube shaking technique will vary among MT → variation in
• Large agglutinated cells are trapped in the tube column for hours reading, grading and interpretation of result
→ (+) : agglutinated RBCs are trapped in the tube column → Stable and well defined end point agglutination reaction that can
be observed or reviewed up to 2-3 days
Three (3) Types of Gel Tests:
• Neutral Gel
→ Does not contain any specific reagent and acts only by its property
of trapping agglutinates
→ Uses: Antibody screening and identification, reverse ABO typing

• Specific Gel
→ Use a specific reagent incorporated into the gel
→ Use: antigen determination

• Gel Low Ionic Antiglobulin Test (GLIAT)

→ Results in separation between the RBCs and the medium without
a washing phase
→ RBCs come in contact with AHG in the upper part of the gel, and
the positive and negative reactions are separated

Immunohematology (Blood Banking), CDU – BSMT3 Page 6 of 9

 REFERENCES
Denise M. Harmening (2012) Modern Blood Banking and Transfusion
Practices 6th Ed.
John Bernard Henry (1996) Clinical Diagnosis and Management by Laboratory
Methods 19th Ed.
Henry’s Clinical Diagnosis and Management by Laboratory Methods 23rd Ed.
Fundamentals of Immunohematology by Mary Louise Turgeon

Different Gel Cards. From the Grifols manufacturer (A-F, left to right)

(A) Gel ABO-Rh card

• Determination of the antigens of the ABO and Rh systems
• Determination of the reverse ABO group

(B) Gel Confirm card

• Confirmation of the blood groups of the ABO and the Rh system.

(C) Gel Coombs card

• For Coombs test
→ Indirect and direct Coombs test
→ Can perform screening and identification of unexpected antibodies, cross-
matching, auto control, and RBC phenotyping.

(D) Neutral Gel card

• Used as a physical medium for saline and enzymatic tests.

(E) Gel Rh pheno + Kell card

• Determination of the antigens of the Rh and Kell systems.

(F) Gel Antibody Screen card

• For Antibody Screening
• Labeled 1, 2, 3, since these will serve as screening cells.

All of these are from Grifols, one of the manufacturers of gel cards used by most
laboratories these days.

• When using a gel card, hemolyzed and grossly icteric blood

samples SHOULD NOT be used.
→ because the results may be difficult to interpret visually.

COMPARISON OF AHG METHODOLOGIES

• To become efficient Transfusion Service departments in the hospital,

one should be able to know which type or which detection methods
should be used to be able to detect clinically significant antibodies in
both direct antiglobulin testing and indirect Coombs test.

DAT METHODS

• There have been numerous studies comparing the tube and gel test
when performing DATs.
• The main difference between the two techniques is that the tube test
requires washing, and the gel test omits a washing stage, resulting in
discrepant results between the two methods.
→ Gell Method: It will omit/no washing stage (washing stage refers
to the washing after the incubation period of the tube test)
▪ Submit to centrifugation immediately.
→ Tube Test: After incubation, for 15 minutes, immediately wash
the test mixture.

IAT METHODS

• LISS and PEG methods are MORE sensitive than the saline tube
method since they are the most labor-intense methods requiring the
most skilled staff.
→ One advantage they have over the gel and solid phase is the
lowered cost of required reagents.
• Gel methods for IAT testing are more sensitive than saline, LISS,
and PEG at detecting passively acquired anti-D in postnatal patients.
• Solid-phase technology can be incorporated as a manual or
automated method for use in IAT.
→ Like gel technology, solid-phase technology is more likely to
detect weakly reactive antibodies than the LISS tube method.

• All blood banks should always be aware of the differences between

these AHG methodologies, especially between the very popular gel
technology and the conventional tube technique.

NEXT PAGE. Outline of the advantage and disadvantages of AHG

methodologies.
Focus more on gel technology and solid phase technology.

Immunohematology (Blood Banking), CDU – BSMT3 Page 7 of 9

Outline of the advantage and disadvantages of AHG methodologies.
Focus more on gel technology and solid phase technology.

Immunohematology (Blood Banking), CDU – BSMT3 Page 8 of 9

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Immunohematology (Blood Banking), CDU – BSMT3 Page 9 of 9